Cyclopropylglycine derivatives and agonists for metabotronic L-glutamate receptors

ABSTRACT

A cyclopropylglycine derivative having the following formula:                    
     and an intermediate compound for the synthesis of the cyclopropylglycine derivative are novel compounds. The cyclopropylglycine derivative shows a selectivity higher than that of the known agonists to metabotropic L-glutamate receptors, and therefore is a metabotropic type agonist to L-glutamate which has excellent characteristics.

This application is a CIP of application Ser. No. 09/214,108 filed Dec.28, 1998 now abandoned.

FIELD OF THE INVENTION

This invention relates to novel cyclopropylglycine derivatives and anagonist comprising the derivative as an active compound. The agonistacts on metabotropic L-glutamate receptors.

BACKGROUND OF THE INVENTION

Glutamate receptors are roughly categorized into two types, namely,ionotropic type (iGluR) and metabotropic type (mGluR). The receptors ofionotropic type (iGluR) are further categorized into NMDA(N-methyl-D-aspartic acid) type and non-NMDA type [Jpn. J.Neuropsychopharmacol., 18(5), 345-365 (1996)].

When NMDA receptors are activated with agonists, they introduce calciumions (Ca²⁺) into cells to increase the intracellular Ca²⁺ concentration.In the cells, accordingly, various Ca²⁺-dependent enzymes are activatedto cause a chain of cellular changes. If the cellular changes proceedover a certain threshold value, the cell is presumed to loseirreversibly its life [Folia Pharmacol. Jpn., No. 104, 177-187 (1994)].

The metabotropic L-glutamate receptors (mGluR) are classified into threegroups (Groups-I, -II and -III) based on their sequence homology,intracellular signal transduction pathway, and selectivity of agonistsfor receptor sub-types. A typical example of the agonist for Group-I isquisqualic acid, which promotes formation of inositol triphosphate (IP₃)and variations of intracellular Ca²⁺ dynamics.

The agonists for Group-II and Group-III inhibit intracellular CAMPformation induced by forskolin. In the agonist selectivity, the mGluR ofGroup-II differs from that of Group-III.

Examples of the agonists for mGluR of Group-II include DCG-IV[(2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine] and L-CCG-I[(2S,1′S,2′S)-2-(2-carboxycyclopropyl)glycine]. Examples of the agonistsfor mGlu R of Group-III include L-AP4 [L-2-amino-4-phosphonobutyricacid] [Japanese Patent Provisional Publications No. 6(1994)-256323 andNo. 6(1994)-24970, and Jpn. J. Neuropsychopharmacol., 18(6), 419-425(1996)].

The agonists for mGluR of Group-II are known to inhibit release oftransmitter at synapses, and consequently to lower the efficiency ofsynaptic conduction. If the synaptic transmission efficiency is loweredin the central nervous system, the muscles in the kinetic system arepresumed to be relaxed. Actually, it has been ascertained by experimentson animals that the agonists for mGluR of Group-II remarkably potentiateanesthesia. These agonists are also known to give sedation (ortranquilizer-like) effect and anti-epileptic effect. Further, sincethese agonists can protect the neurons cells from death in vivo and invitro caused by excitatory amino acids, they are expected to be used asneuron protectors. Since the agonists for mGluR of Group-II are utterlynew agonists for glutamate receptors and seem to be indispensable forpharmaceutical studies of the central nervous system, they are of greatvalue as reagents for the laboratory study. [Folia Pharmacol. Jpn., No.104, 177-187 (1994)].

DCG-IV (which is one of the known agonists for mGluR of Group-II)strongly activates the mGluR, and is hence expected to act as a neuronprotector. However, since DCG-IV also activates NMDA receptors (whichare presumed to be concerned with cell death caused by excitatory aminoacids), it is desired to develop a new agonist for mGluR having no NMDAactivating component.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a novelcyclopropylglycine derivative having better selectivity andcharacteristics than known agonists for glutamic acid receptors ofmetabolic regulation type.

It is another object of the invention to provide a new compoundemployable as an intermediate for preparing the novel cyclopropylglycinederivative.

This invention resides in a cyclopropylglycine derivative having thefollowing formula (I).

In the formula (I), each of R¹ and R² independently represents ahydroxyl group or an alkoxy group having 1 to 6 carbon atoms, each of R³and R⁴ independently represents a hydrogen atom or an alkyl group having1 to 6 carbon atoms, and each of X¹ and X² independently represents ahalogen atom.

The invention also resides in an agonist which acts on L-glutamic acidreceptors of metabolic regulation type and which comprises thecyclopropylglycine derivative having the formula (I) as an activecomponent.

Further, the invention resides in a pharmaceutical compositioncomprising the cyclopropylglycine derivative having the formula (I) asan active component.

The intermediate provided by the invention is a lactam derivative havingthe following formula (II).

In the formula (II), R² represents a hydroxyl group or an alkoxy grouphaving 1 to 6 carbon atoms, R³ represents a hydrogen atom or an alkylgroup having 1 to 6 carbon atoms, and each of X¹ and X² independentlyrepresents a halogen atom.

PREFERRED EMBODIMENTS OF THE INVENTION

The cyclopropylglycine derivative of the invention has the followingformula (I):

In the formula (I), each of R¹ and R² independently represents ahydroxyl group or an alkoxy group having 1 to 6 carbon atoms (e.g.,methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, and isobutoxy), eachof R³ and R⁴ independently represents a hydrogen atom or an alkyl grouphaving 1 to 6 carbon atoms (e.g., methyl, ethyl, n-propyl, isopropyl,n-butyl, and isobutyl), and each of X¹ and X² independently represents ahalogen atom (e.g., fluorine, chlorine, bromine, and iodine).

The cyclopropylglycine derivative of the invention can be used in theform of a free acid or its salt. Preferably, in the formula (I), each ofR¹ and R² represents a hydroxyl group, each of R³ and R⁴ represents ahydrogen atom, and each of X¹ and X² represents a fluorine atom. Inother words, a preferred embodiment of the derivative is2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine. This compound has thefollowing eight optical isomers.

L-I: (2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

L-II: (2S,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

L-III: (2S,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

L-IV: (2S,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

D-I: (2R,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

D-II: (2R,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

D-III: (2R,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

D-IV: (2R,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine,

Most preferred is(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-I].

The cyclopropylglycine derivative of the invention can be prepared inthe manner shown in the following reaction scheme-1 and -2. In thereaction scheme-1, a known olefin compound [E-1 or Z-1; see J. Org.Chem., 59(1), 97-103(1994)] is caused to react with sodiumchlorodifluoroacetate to prepare two optical isomers of2-(2′-benzyloxymethyl-3′,3′-difluoro)cyclopropylethyleneglycol1,2-O-acetonide [reaction from E-1 is described in Tetrahedron:Asymmetry, 5(8), 1423-1426(1994)]. From thus obtained compounds, thetarget cyclopropylglycine derivatives can be prepared in the mannershown in the reaction scheme-2. The reaction scheme-2 shows, forexample, the reactions for preparing the derivatives of L-I and D-II.

The preparation examples of the cyclopropylglycine derivatives of theinvention are described below. In the manners of the following examples,the derivatives other than those mentioned below can be easily alsoprepared.

EXAMPLE 1 Preparation of [(2S,1′R,2′S)-2] and [(2S,1′S,2′R)-2] from[Z-1]—Reaction scheme-1

A diethyleneglycol dimethyl ether solution containing 7.5 g of(Z,4′S)-3-benzyloxy-1-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1-propene[Z-1] was heated to 180° C. Into the heated solution, a diethyleneglycoldimethyl ether solution containing 45.8 g of sodiumchlorodifluoroacetate was dropwise added for 10 hours. The resultingsolution was heated at 180° C. for 1 hour, and then cooled. After anice-water mixture was added, the solution was extracted with hexane. Theextracted portion was washed with water, dried over magnesium sulfate,and concentrated under reduced pressure. The obtained residue wasroughly purified by silica gel column chromatography (hexane: ethylacetate=30:1, volume ratio), and then the optical isomers were separatedby medium pressure chromatography (hexane:ethyl acetate=4:1, volumeratio), to obtain 2.6 g and 5.6 g, respectively, of the followingproducts of [(2S,1′R,2′S)-2] and [(2S,1′S,2′R)-2].

[(2S,1′R,2′S)-2]: colorless oil

[α]_(D) ^(27.6)=−37.6 (c 0.978, CHCl₃)

[(2S,1′S,2′R)-2] : colorless oil

[α]_(D) ^(28.0)=−35.6 (c 0.999, CHCl₃)

EXAMPLE 2 Preparation of [(2S,1′S,2′S)-2] and [(2S,1′R,2′R)-2] from[E-1] —Reaction scheme-1

The procedure of Example 1 was repeated except for using 1.3 g of(E,4′S)-3-benzyloxy-1-(2′,2′-dimethyl-1′,3′-dioxolan-4′-yl)-1-propene[E-1] as the starting material, to prepare 0.76 g and 0.45 g,respectively, of the following products of [(2S,1′S, 2′S)-2] and[(2S,1′R,2′R)-2].

[(2S,1′S,2′S)-2]: colorless oil

[α]_(D) ^(26.0)=−20.9 (c 1.00, CHCl₃)

[(2S,1′R,2′R)-2] : colorless oil

[α]_(D) ^(24.8)=+28.7 (c 1.06, CHCl₃)

EXAMPLE 3 Preparation of(2R,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [D-II] andits esters—Reaction scheme-3

(1) Preparation of [(2S,1′S,2′S)-3] from [(2S,1′S,2′S)-2]

Into 2 mL of a methanol solution containing 300 mg of [(2S,1′S,2′S)-2],0.5 mL of 10% hydrochloric acid was dropwise added and caused to reactfor 6 hours at room temperature. The reaction mixture was concentratedunder reduced pressure. After an aqueous saturated sodiumhydrogencarbonate solution was added, the resultant solution wasextracted with ethyl acetate. The organic portion was collected, washedwith an aqueous saturated sodium chloride solution, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure.

The residue was dissolved in 2 mL of N,N-dimethylformamide. Under argonatmosphere, 138.0 mg of imidazole was added to the solution, and then167.0 mg of t-butyldimethylsilyl chloride (TBDMS-C1) was added at 0° C.,followed by stirring for 5 hours at room temperature. After an aqueoussaturated ammonium chloride solution was added, the solution wasextracted with diethyl ether. The organic portion was collected, washedwith an aqueous saturated sodium chloride solution, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. Theresultant residue was purified by silica gel column chromatography(hexane: ethyl acetate =15:1, volume ratio). From the eluent, 302.0 mgof the following product of [(2S,1′S, 2′S)-3] was obtained.

[(2S,1′S,2′S)-3]: colorless oil

[α]_(D) ^(28.4)=−10.6 (c 1.00, CHCl₃)

(2) Preparation of [(2R,1′S,2′S)-4] from [(2S,1′S,2′S)-3]

Under argon atmosphere, 0.12 mL of diphenylphosphoryl azide and 87 μL ofdiethyl azodicarboxylate were added at 0° C. to 2 mL of atetrahydrofuran solution containing 136 mg of [(2S,1′S,2′S)-3] and 165mg of triphenylphosphine, and then the mixture was stirred for 6 hoursat room temperature. To the resulting solution, an aqueous saturatedsodium chloride solution was added. Then, the solution was extractedwith diethyl ether;

The ether portion was collected, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (hexane:ethyl acetate=15:1, volumeratio). From the eluent, 92 mg of the following product of[(2R,1′S,2′S)-4] was obtained.

[(2R,1′S,2′S)-4]: colorless oil

[α]_(D) ^(26.8)=−3.03 (c 0.99, CHCl₃)

(3) Preparation of [(2R,1′S,2′S)-5] from [(2R,1′S, 2′S)-4]

To 3 mL of a tetrahydrofuran solution containing 92 mg of[(2R,1′S,2′S)-4], tetrabutylammonium fluoride was added at 0° C. Theresulting solution was stirred for 3 hours. After an aqueous saturatedsodium chloride solution was added, the solution was extracted withdiethyl ether.

The ether portion was collected, dried over anhydrous magnesium sulfate,and concentrated under reduced pressure. The residue was purified bysilica gel column chromatography (hexane:ethyl acetate=10:1, volumeratio). From the eluent, 65 mg of the following product of[(2R,1′S,2′S)-5] was obtained.

[(2R,1′S,2′S)-5]: colorless oil

[α]_(D) ^(26.0)=−42.4 (c 0.97, CHCl₃)

(4) Preparation of [(2R,1′S,2′S)-6] from [(2R,1′S,2′S)-5]

Under hydrogen atmosphere, 2 mL of an ethyl acetate suspensioncontaining 2.1 g of [(2R,1′S,2′S)-5] and 5% Pd/C(charcoal) was stirredfor 3 hours at room temperature under atmospheric pressure. The reactionmixture was filtered, and then concentrated under reduced pressure. Theresidue was dissolved in 18 mL of a mixture solvent of dioxane/water(2/1, volume ratio). To the solution, 3.1 g of sodium hydrogencarbonateand 3.5 g of di-t-butyl dicarbonate (Boc₂O) were added. The resultingmixture was stirred for 1 hour at room temperature. After an aqueoussaturated sodium chloride solution was added, the solution was extractedwith ethyl acetate. The organic portion was collected, dried overanhydrous magnesium sulfate, and concentrated under reduced pressure.

The residue was purified by silica gel column chromatography(hexane:ethyl acetate=3:2, volume ratio). From the eluent, 3.3 g of thefollowing product of [(2R,1′S,2′S)-6] was obtained.

[(2R,1′S,2′S)-6]: colorless prisms

m.p.: 101.5-103.5° C.

[α]_(D) ^(29.6)=−19.99 (C 1.01, CHCl₃)

(5) Preparation of [(2R,1′S,2′S)-7] from [(2R,1′S,2′S)-6]

To 20 mL of an acetone solution containing 500 mg of [(2R,1′S,2′S)-6], 5mL of Jones' reagent was added under chilling with ice. After one hour,3 mL of isopropyl alcohol was added. The mixture was stirred for 30minutes, and then water was added. The resulting solution was extractedwith ethyl acetate, and then the organic portion was collected, driedover anhydrous magnesium sulfate, and concentrated under reducedpressure.

The residue was dissolved in 5 mL of diethyl ether, and then an ethersolution containing diazomethane was added under chilling with ice. Thereaction mixture was concentrated under reduced pressure, and theobtained residue was purified by silica gel column chromatography(hexane:ethyl acetate=5:1, volume ratio). From the eluent, 442 mg of thefollowing product of [(2R,1′S,2′S)-7] was obtained.

[(2R,1′S,2′S)-7]: colorless prisms

m.p.: 59.0-61.0° C.

[α]_(D) ^(29.2)=−34.84 (c 0.528, CHCl₃)

(6) Preparation of [(2R,1′S,2′S)-8] from [(2R,1′S,2′S)-7]

Under hydrogen atmosphere, 1 mL of an ethyl acetate suspensioncontaining 1.0 g of [(2R,1′S,2′S)-7] and palladium hydroxide was stirredfor 5 hours at room temperature under atmospheric pressure. The reactionmixture was filtered, and concentrated under reduced pressure. Theresidue was treated in the #nner described in the (5) above, and thenpurified by silica gel column chromatography (hexane:ethyl acetate=1:1,volume ratio). The eluent was dissolved in 5 mL of benzyl alcohol, andthen 0.87 mL of titanium tetraisopropoxide was added. After stirring for3 hours at 70° C., the resulting solution was purified by silica gelcolumn chromatography (hexane:ethyl acetate=20:1, volume ratio). Theeluent was further purified by medium pressure chromatography(hexane:ethyl acetate=4:1, volume ratio), to prepare 393.0 mg of thefollowing product of [(2R,1′S,2′S)-8].

[(2R,1′S,2′S)-8]: colorless prisms

m.p.: 114.5-116.5° C.

[α]_(D) ^(28.0)=+12.12 (c 1.006, CHCl₃)

(7) Preparation of [(2R,1′S,2′S)-9] from [(2R,1′S,2′S)-8]

Under hydrogen atmosphere, 0.5 mL of a methanol suspension containing100 mg of [(2R,1′S,2′S)-8] and palladium hydroxide was stirred for 3hours. The reaction mixture was filtered, and concentrated under reducedpressure. The residue was dissolved in 1.5 mL of a mixture solvent oftetrahydrofuran/water (1/1, vol/vol), and then 50 μL of 36% hydrochloricacid was added. Subsequently, the solution was stirred for 2 hours at50° C.

The reaction mixture was concentrated under reduced pressure, and thenpurified by HPLC (H₂O) using a reversed phase column (ODS column). Thus,18.7 mg of the following product of [(2R,1′S,2′S)-9] [hydrochloride of(2R,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)-cyclopropylglycine (D-II)pwas obtained.

[(2R,1′S,2′S)-9]=hydrochloride of D-II

colorless prisms

m.p.: 160° C. (decomposed)

[α]_(D) ^(25.6)=−38.54 (c 0.890, H₂O)

IR (KBr) ν cm⁻¹: 3447, 3034, 2924, 1717, 1637, 1398, 1239, 1028, 948,792.

¹H-NMR (400 MHz, D₂O)δ: 2.73 (1H, ddd, J=11.4, 10.9, 7.3 Hz), 2.97 (1H,dd, J=14.1, 7.2 Hz), 3.82 (1H, d, J=10.9 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 30.56 (dd, J=10.2, 9.3 Hz), 33.84 (dd,J=11.1, 10.9 Hz), 53.05, 113.08 (dd, J=292.3, 285.7 Hz), 171.89, 173.29.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.72 (1F, dd, J=158.2, 11.8 Hz), −70.05(1F, dd, J=159.8, 13.7 Hz).

(8) Preparation of [(2R,1′S,2′S)-10] from [(2R,1′S,2′S)-7]

74.8 mg of [(2R,1′S,2′S)-7] was treated in the manner described in theabove (6) for eliminating benzyl ether and performing Jones' oxidation.The obtained residue was dissolved in a mixture solvent of 10%hydrochloric acid/tetrahydrofuran (1/1 vol/vol), and then the solutionwas stirred for 1 hour at 50° C.

The reaction mixture was concentrated under reduced pressure, and thenpurified by HPLC (H₂O) using a reversed phase column (ODS column). Thus,14.5 mg of the following product of [(2R,1′S,2′S)-10] (hydrochloric saltof monomethyl ester of(2R,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine (D-II)] wasobtained.

[(2R,1′S,2′S)-10]=hydrochloride of monomethyl

ester of D-II:

colorless prisms

[α]_(D) ^(26.0)=−31.99 (c 1.45, H₂O)

¹H-NMR (300 MHz, D₂O)δ: 2.73 (1H, ddd, J=18.4, 11.4, 1.7 Hz), 2.97 (1H,dd, J=14.4, 7.5 Hz), 3.88 (3H, s), 4.14 (1H, d, J=11.0 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 29.78 (dd, J=11.5, 8.4 Hz), 33.87 (dd,J=10.9, 10.9 Hz), 52.24, 57.15, 112.86 (dd, J=292.1, 286.0 Hz), 171.05,171.79.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.70 (1F, dd, J=160.6, 9.3 Hz), −70.01(1F,dd, J=157.9, 14.0 Hz).

EXAMPLE 4 Preparation of(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-1] andits esters—Reaction scheme-4

(1) Preparation of [(2R,1′S,2′S)-11] from [(2S,1′S,2′S)-3]

To 10 mL of a tetrahydrofuran solution containing 3.0 g of[(2S,1′S,2′S)-3] and 4.0 g of triphenylphosphine, 2.1 mL of diethylazodicarboxylate and 1.6 g of benzoic acid were added under chillingwith ice. The mixture was then stirred for 2 hours at room temperature.After an aqueous saturated sodium chloride solution was added, thesolution was extracted with diethyl ether. The organic portion wascollected, dried over anhydrous sodium sulfate, and concentrated underreduced pressure.

The residue was purified by silica gel column chromatography(hexane:ethyl acetate=30:1, volume ratio). From the eluent, 3.5 g of thefollowing product of [(2R,1′S,2′S)-11] was obtained.

[(2R,1′S,2′S)-11]: colorless oil

[α]_(D) ^(25.6)=+3.00 (c 1.000, CHCl₃)

(2) Preparation of [(2R,1′S,2′S)-3] from [(2R,1′S,2′S)-11]

5 mL of an aqueous 1N-NaOH solution was added to 10 mL of a methanolsolution containing 3.3 g of [(2R,1′S, 2′S)-11], and the resultingsolution was stirred for 6 hours at room temperature. The solution wasthen concentrated under reduced pressure. After water was added to theresidue, the solution was extracted with diethyl ether.

The organic portion was collected, dried over anhydrous magnesiumsulfate, and concentrated under reduced pressure. The residue waspurified by silica gel column chromatography (hexane:ethyl acetate=15:1,volume ratio). From the eluent, 1.7 g of the following product of[(2R,1′S,2′S)-3] was obtained.

[(2R,1′S,2′S)-3]: colorless oil

[α]_(D) ^(24.4)=−16.89 (c 1.006, CHCl₃)

(3) Preparation of [(2S,1′S,2′S)-9] from [(2R,1′S, 2′S)-3]

The [(2R,1′S,2′S)-3] obtained in the (2) above was treated in themanners described in the (2) to (7) of Example 3, to prepare thefollowing product of [(2S,1′S,2′S)-9] (hydrochloride of(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-I]).

[(2S,1′S,2′S)-9]=hydrochloride of L-I:

colorless prisms

m.p.: 155° C. (decomposed)

[α]_(D) ^(24.0)=+50.64 (c 1.398, H₂O)

IR (KBr) ν cm⁻¹: 3414, 3061, 1712, 1616, 1477, 1226, 1040, 737, 620.

¹H-NMR (400 MHz, D₂O)δ: 2.78 (1H, ddd, J=13.0, 10.8, 7.5 Hz), 2.86 (1H,dd, J=14.2, 7.5 Hz), 3.92 (1H, d, J=10.7 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 30.88 (dd, J=12.0, 8.9 Hz), 33.29 (dd,J=11.1, 10.9 Hz), 53.20, 113.26 (dd, J=289.6, 286.3 Hz), 171.77, 172.89.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −70.35 (1F, dd, J=157.9, 13.1 Hz), −67.51(1F, dd, J=156.9, 12.8 Hz).

(4) Preparation of [(2S,1′S,2′S)-10] from [(2R,1′S,2′S)-3]

The [(2R,1′S,2′S)-3] obtained in the (2) above was treated in the mannerdescribed in the (8) of Example 3, to prepare the following product of[(2S,1′S,2′S)-10] (hydrochloride of monomethyl ester of(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-I]).

[(2S,1′S,2′S)-10]=hydrochloride of monomethyl

ester of L-I:

colorless prisms

[α]_(D) ^(24.8)=+52.88 (c 1.49, H₂O)

¹H-NMR (300 MHz, D₂O)δ: 2.60-2.80 (2H, m), 3.91 (3H, s), 4.16 (1H, d,J=10.7 Hz).

¹³C-NMR (75.5 MHz, D₂O)δ: 29.94 (dd, J=13.0, 8.3 Hz), 33.38 (dd, J=11.1,10.8 Hz), 53.39, 57.08, 112.73 (dd, J=290.0, 286.2 Hz), 170.61, 171.43.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −70.65 (1F, dd, J=160.7, 13.0 Hz), −67.81(1F, dd, J=157.4, 13.4 Hz).

EXAMPLE 5 Preparation of(2R,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine[D-1]—Reaction scheme-5

(1) Preparation of [(2R,1′R,2′R)-9] from [(2S,1′R,2′R)-2]

[(2S,1′R,2′R)-2] was treated in the manners described in the (1) to (7)of Example 3, to prepare the following product of [(2R,1′R,2′R)-9](hydrochloride of(2R,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [D-I]).

[(2R,1′R,2′R)-9]=hydrochloride of D-I:

colorless prisms

m.p.: 153° C. (decomposed)

[α]_(D) ^(27.6)=−47.49 (c 0.88, H₂O)

IR (KBr) ν cm⁻¹: 3424, 3035, 1721, 1638, 1477, 1212, 1045, 738, 622.

¹H-NMR (400 MHz, D₂O)δ: 2.72 (1H, ddd, J=13.2, 10.8, 7.6 Hz), 2.80 (1H,dd, J=14.4, 7.5 Hz), 3.81 (1H, d, J=10.7 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 31.24 (dd, J=11.8, 8.9 Hz), 33.78 (brt),53.76, 113.52 (dd, J=289.0, 286.0 Hz), 172.37, 173.58.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −70.11 (1F, dd, J=157.4, 13.3 Hz), −67.44(1F, dd, J=157.4, 15.9 Hz).

EXAMPLE 6 Preparation of(2S,1′R,2′R)-2-(2′-carboxy-3′,3′-di-fluoro)cyclopropylglycine [L-II] andits esters—Reaction scheme-6

(1) Preparation of [(2S,1′R,2′R)-9] from [(2R,1′R,2′R)-11]

In the manner described in Example 4, the following product of[(2S,1′R,2′R)-9] (hydrochloride of (2S,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-III) wasprepared.

[(2S,1′R,2′R)-9]=hydrochloride of L-II:

colorless prisms

m.p.: 165° C. (decomposed)

[α]_(D) ^(24.0)=+40.68 (c 0.870, H₂O)

IR (KBr) ν cm⁻¹: 3209, 3046, 2928, 1716, 1646, 1387, 1226, 1028, 950,784.

¹H-NMR (400 MHz, D₂O)δ: 2.67 (1H, dddd, J=11.6, 10.9, 7.3, 1.6 Hz), 2.90(1H, dd, J=14.3, 7.3 Hz), 3.72 (1H, d, J=10.9 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 30.66 (dd, J=10.0, 9.5 Hz), 34.12 (dd,J=10.8, 10.6 Hz), 53.29, 113.21 (dd, J=291.9, 285.7 Hz), 172.21, 173.64.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.73 (1F, dd, J=160.8, 12.7 Hz), −70.07(1F, dd, J=159.7, 13.6 Hz).

(2) Preparation of [(2S,1′R,2′R)-10] from [(2R,1′R,2′R)-7]

In the manner described in Example 4, the following product of[(2S,1′R,2′R)-10] (hydrochloride of monomethyl ester of(2S,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-II]) wasprepared.

[(2S,1′R,2′R)-9]=hydrochloride of monomethyl

ester of L-II:

colorless prisms

[α]_(D) ^(25.6)=+50.67 (c 1.03, H₂O)

¹H-NMR (300 MHz, D₂O)δ: 2.81 (1H, ddd, J=18.4, 11.3, 1.7 Hz), 3.07 (1H,dd, J=14.0. 7.1 Hz), 3.91 (3H, s), 4.20 (1H, J=11.3 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 29.78 (dd, J=11.2, 9.0 Hz), 33.99 (br),52.22, 57.14, 112.93 (dd, J=292.2, 286.0 Hz), 171.04, 171.77.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.70 (1F, dd, J=157.6, 9.5 Hz), −70.01 (1F,dd, J=160.1, 13.8 Hz).

(3) Preparation of [(2S,1′R,2′R)-12] from [(2R,1′R,2′R)-9]

10 mg of the [(2R,1′R,2′R)-9] obtained in the above (1) was dissolved ina mixture solvent of diethyl ether/methanol (1/1, vol/vol), and then anether solution containing diazomethane was added. After the resultingsolution was concentrated under reduced pressure, 10% hydrochloric acidwas added. The obtained mixture was again concentrated under reducedpressure, to prepare 11.2 mg of the following product of[(2S,1′R,2′R)-12] (hydrochloride of dimethyl ester of(2S,1′R,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-II]).

[(2S,1′R,2′R)-12]=hydrochloride of dimethyl

ester of L-II:

colorless prismatic crystals

¹H-NMR (400 MHz, D₂O)δ: 2.88 (1H, dd, J=18.6, 11.3 Hz), 3.18 (1H, dd,J=13.6, 7.3 Hz), 3.83 (3H, s), 3.90 (3H, s), 4.21 (1H, J=11.1 Hz).

¹³C-NMR (75.5 MHz, D₂O)δ: 29.56 (dd, J=8.9, 8.5 Hz), 33.07 (dd, J=12.5,10.2 Hz), 51.99, 56.29, 57.02, 112.90 (dd, J=292.0, 286.6 Hz), 170.13,170.71.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.80 (1F, dd, J=161.1, 12.9 Hz), −70.15(1F, dd, J=160.7, 13.1 Hz).

EXAMPLE 7 Preparation of(2R,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine[D-III]—Reaction scheme-7

(1) Preparation of [(2R,1′R,2′S)-7] from [(2S,1′R,2′S)-2]

[(2S,1′R,2′S)-2] was treated in the manner described in Example 3, toprepare [(2R,1′R,2′S)-7] as a colorless oil.

(2) Preparation of [(3S,4R,5R)-13] from [(2R,1′R,2′S)-7]

Under hydrogen atmosphere, 1 mL of a methanol suspension containing 125mg of [(2R,1′R,2′S)-7] and 5% Pd/C was stirred for 2 hours at roomtemperature under atmospheric pressure. The reaction mixture wasfiltered, and then concentrated under reduced pressure. The residue wasdissolved in 5 mL of acetone, and then Jones' reagent was added. Afterstirring for 30 minutes, isopropyl alcohol was dropwise added. Thesolution was further stirred for 30 minutes, and an aqueous saturatedsodium chloride solution was added. The resultant solution was extractedwith diethyl ether, and the organic portion was collected, dried overanhydrous magnesium sulfate, and concentrated under reduced pressure.The residue was purified by silica gel column chromatography(hexane:ethyl acetate=5:1, volume ratio). From the eluent, 60 mg of((3S,4R,5R)-13] was obtained as a colorless oil.

(3) Preparation of [(3S,4R,5R)-14] and [(2R,1′R,2′S)-9] from[(3S,4R,5R)-13]

4 mL of 1N hydrochloric acid was added to 33 mg of [(3S,4R,5R)-13], andthe mixture was stirred for 5 hours at 60° C. The reaction mixture wasconcentrated under reduced pressure, and then purified by HPLC (H₂O)using a reversed phase column (ODS column). Thus, 7.9 mg and 7.6 mg,respectively, of the following products of [(3S,4R,5R)-14] and [(2R,1′R,2′S)-9] (hydrochloride of(2R,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [D-III]were obtained.

[(3S,4R,5R)-14] : colorless prisms

¹H-NMR (400 MHz, D₂O)δ: 3.00 (3H, dd, J=11.7, 8.2 Hz), 3.14 (1H, dd,J=10.9, 8.2 Hz), 4.58 (1H, s).

¹³C-NMR (100.6 MHz, D₂O)δ: 30.18 (dd, J=12.9, 12.8 Hz), 32.84 (dd,J=13.7, 13.4 Hz), 56.81, 112.11 (dd, J=292.5, 278.2 Hz), 175.15, 176.05.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −84.26 (1F, d, J=157.1 Hz), −64.32 (1F, ddd,J=157.0, 12.6, 9.2 2 Hz).

[(2R,1′R,2′S)-9]=hydrochloride of D-III:

colorless prisms

[α]_(D) ^(25.0)+17.34 (c 0.830, H₂O)

IR (KBr) ν cm⁻¹: 3422, 3038, 1721, 1627, 1471, 1400, 1225, 1045, 975,710.

¹H-NMR (300 MHz, D₂O)δ: 2.61 (1H, ddd, J=11.8, 11.7, 11.6 Hz), 3.01 (1H,dd, J=12.6, 11.4 Hz), 4.55 (1H, d, J=11.3 Hz).

¹³C-NMR (75.5 MHz, D₂O)δ: 29.12 (dd, J=11.7, 8.9 Hz), 31.25 (dd, J=10.6,8.0 Hz), 50.46, 112.5 (dd, J=292.9, 282.9 Hz), 171.60, 173.44.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −79.04 (1F, d, J=159.1 Hz), −59.05 (1F, ddd,J=157.0, 13.1, 1 3.0 Hz).

EXAMPLE 8 Preparation of (2S,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-IV]—Reaction scheme-8

(1) Preparation of [(2S,1′R,2′S)-7] from [(2S,1′R,2′S)-2]

[(2S,1′R,2′S)-2] was treated in the manner described in Example 4, toprepare [(2S,1′R,2′S)-7] as a colorless prisms.

(2) Preparation of [(3S,4R,5S)-13] from [(2S,1′R,2′S)-7]

[(2S,1′R,2′S)-7] was treated in the manner described in Example 7, toprepare [(3S,4R,5S)-13] as a colorless prisms.

(3) Preparation of [(3S,4R,5S)-14] and [(2S,1′R,2′S)-9] from[(3S,4R,5S)-13]

[(3S,4R,5S)-13] was treated in the manner described in Example 7, toprepare the following products of [(3S, 4R,5S)-14] and [(2S,1′R,2′S)-9](hydrochloride of(2S,1′R,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-IV]).

[(3S,4R,5S)-14]: colorless prisms

¹H-NMR (400 MHz, D₂O)δ: 2.99 (1H, dd, J=11.8, 8.5 Hz), 3.17 (1H, ddd,J=10.6, 8.1, 7.9 Hz), 4.93 (1H, dd, J=7.4, 1.8 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 28.59 (dd, J=12.5, 12.4 Hz), 33.83 (dd,J=13.42, 13.35 Hz), 57.85, 112.68 (dd, J=293.0, 279.5 Hz), 174.46,175.72.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −81.75 (1F, d, J=161.1 Hz), −63.03 (1F, ddd,J=161.0, 12.0, 9.4 Hz).

[(2S,1′R,2′S)-9]=hydrochloride of L-IV:

colorless prisms

[α]_(D) ^(26.5)=+70.78 (c 0.955, H₂O)

IR (KBr) ν cm⁻¹: 3455, 3032, 1732, 1626, 1472, 1219, 1156, 1085, 1046,975.

¹H-NMR (300 MHz, D₂O)δ: 2.71 (1H, ddd, J=10.5, 10.4, 9.4 Hz), 3.05 (1H,dd, J=12.0, 11.9 Hz), 4.71 (1H, d, J=11.5 Hz).

¹³C-NMR (75.5 MHz, D₂O)δ: 29.10 (dd, J=10.1, 9.9 Hz), 31.07 (dd, J=11.6,9.0 Hz), 48.76, 112.51 (dd, J=292.4, 285.3 Hz), 171.73, 172.64.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −83.27 (1F, d, J=161.3 Hz), −59.10 (1F, ddd,J=161.2, 10.7, 9.3 Hz).

EXAMPLE 9 Preparation of(2R,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine[D-IV]—Reaction scheme-9

In the manner described in Example 7, the following products of[(3R,4S,5R)-14] and [(2R,1′S,2′R)-9] (hydrochloride of(2R,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [D-IV])were prepared.

[(3R,4S,5R)-14]: colorless prisms

¹H-NMR (400 MHz, D₂O)δ: 2.99 (1H, dd, J=11.9, 8.6 Hz), 3.17 (1H, dd,J=18.7, 8.1 Hz), 4.58 (1H, d, J=7.3 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 28.57 (dd, J=12.5, 12.4 Hz), 33.82 (dd,J=13.4, 13.3 Hz), 57.83, 112.67 (dd, J=293.0, 279.4 Hz), 174.44, 175.70.

[(2R,1′S,2′R)-9] =hydrochloride of D-IV:

colorless prisms

[α]_(D) ^(22.4)=−67.99 (c 1.050, H₂O)

IR (KBr) ν cm⁻¹: 3432, 3039, 1731, 1636, 1471, 1219, 1155, 1084, 1047,974.

¹H-NMR (400 MHz, D₂O)δ: 2.65 (1H, dddd, J=11.8, 11.7, 10.1, 2.5 Hz),3.06 (1H, ddd, J=12.2, 12.1, 2.2 Hz), 4.58 (1H, d, J=11.4 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 29.27 (dd, J=10.3, 9.6 Hz), 31.50 (brt),49.54 112.66 (dd, J=291.8, 283.9 Hz), 171.78, 173.25.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −83.24 (1F, d, J=161.4 Hz), −59.14 (1F, ddd,J=161.4, 11.7, 9.9 Hz).

EXAMPLE 10 Preparation of (2S,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-III] —Reaction scheme-10

In the manner described in Example 8, the following products of[(3R,4S,5S)-14] and [(2S,1′S,2′R)-9] (hydrochloride of(2S,1′S,2′R)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-III])were prepared.

[(3R,4S,5S)-14] : colorless prisms

¹H-NMR (400 MHz, D₂O)δ: 3.00 (1H, dd, J=11.7, 8.2 Hz), 3.14 (1H, dd,J=10.9, 8.3 Hz), 4.57 (1H, s).

¹³C-NMR (100.6 MHz, D₂O)δ: 30.19 (dd, J=12.9, 12.6 Hz), 32.84 (t, J=13.4Hz), 56.81, 112.05 (t, J=291.8 Hz), 175.16, 176.06.

[(2S,1′S,2′R)-9] =hydrochloride of L-III:

colorless prisms

[α]_(D) ^(25.4)=−13.90 (c 0.820, H₂O)

IR (KBr) ν cm⁻¹: 3438, 3033, 1720, 1637, 1471, 1399, 1224, 1043, 974,708.

¹H-NMR (300 MHz, D₂O)δ: 2.58 (1H, ddd, J=11.7, 11.5, 11.3 Hz), 2.99 (1H,dd, J=12.2, 10.7 Hz), 4.51 (1H, d, J=11.5 Hz).

¹³C-NMR (75.5 MHz, D₂O)δ: 29.37 (dd, J=10.9, 9.5 Hz), 31.55 (dd, J=10.8,10.2 Hz), 50.65, 112.77 (dd, J=292.7, 282.9 Hz), 171.81, 173.63.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −79.02 (1F, d, J=158.9 Hz), −59.03 (1F, ddd,J=157.0, 13.1, 12.5 Hz).

EXAMPLE 11 Preparation of(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine[L-I]—Reaction scheme-11

(1) Preparation of (4S)-3-[(E)-4′-bromo-4′,4′-difluoro-2′-butenoyl]-4-benzyl-2-oxazolidinone (Compound-5a)

Under argon atmosphere, 15 mL of an acetonitrile solution containing 3.7g of (4S)-N-bromoacetyl-4-benzyl-2-oxazolidinone and 3.6 g oftriphenylphosphine was stirred for two days at 50° C. After adding 6.5mL of 2N-NaOH aqueous solution, the reaction mixture was extracted withethyl ether. The organic portion was collected, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure to obtaincrude phosphorane-9a.

Independently, a mixture of 1.7 mL of ethyl bromodifluoroacetate and14.7 mL of 0.93M DIBAL-H (diisobutylaluminum hydride hexane solution) in10 mL of ethyl ether was stirred for 20 minutes at −78° C. To themixture, 5 mL of methanol and 10 mL of 5% aqueous HCl were successivelyadded. The resultant mixture was stirred for 10 minutes at roomtemperature, and then extracted with ethyl ether. The organic portionwas collected, washed successively with an aqueous saturated sodiumhydrogencarbonate solution and an aqueous saturated sodium chloridesolution, dried over anhydrous magnesium sulfate, and concentrated underreduced pressure.

The residue and crude phosphorane 9a were dissolved in 38 mL oftetrahydrofuran (THF), and thus prepared solution was stirred for 5hours at room temperature. Subsequently, the solvent was distilled offunder reduced pressure, and the residue was purified by silica gelcolumn chromatography to obtain 2.88 of(4S)-3-[(E)-4′-bromo-4′,4′-difluoro-2′-butenoyl]-4-benzyl-2-oxazolidinone(Compound-5a) as a colorless oil.

(2) Preparation of (2S,1′S,2′S,4″S)-ethylN-diphenylmethylidene-2-[2′-{(4″-benzyl-2″-oxazolidinon-3″-yl)-carbonyl}-3′,3′-difluoro)cyclopropylglycinate(Compound-7a)

Under argon atmosphere, a mixture of 756 mg of ethylN-(diphenylmethylidene)glycinate and LDA (lithium diisopropylamide),prepared from 0.47 mL of N,N-diisopropylamine and 1.87 mL of 1.65 Mn-butyl lithium hexane solution, in 15 mL of dimethylformamide (DMF) wasstirred for 15 minutes at −20° C. Independently, 926 mg of theabove-prepared(4S)-3-[(E)-4′-bromo-4′,4′-difluoro-2′-butenoyl]-4-benzyl-2-oxazolidinone(Compound-5a) was dissolved in 10 mL, of DMF, and the obtained solutionwas added to the mixture. The resulting mixture was stirred for 2 hoursat the same temperature. After adding an aqueous saturated ammoniumchloride solution to quench the reaction, the reaction mixture wasextracted with ethyl ether. The organic portion was collected, washedwith an aqueous saturated sodium chloride solution, dried over anhydrousmagnesium sulfate, and concentrated under reduced pressure. The residuewas purified by silica gel column chromatography (hexane:ethylacetate=7:1, volume ratio), to prepare 758 mg of Compound-7a (colorlessoil, yield: 54%) and 245 mg of Compound-8a (colorless oil, yield: 16%)

(3) Preparation of (2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-I]

Under reduced pressure (40 mmHg), a mixture of 1 mL of benzyl alcoholand 154 mg of Ti(O-i-Pr)₄ was stirred for 30 minutes at roomtemperature. The obtained liquid and 100 mg of Compound-7a preparedabove were mixed and stirred for 7 hours at 70° C. The reaction mixturewas purified by silica gel column chromatography (hexane: ethylacetate=10:1, volume ratio), to obtain 87 mg of dibenzyl ester-11(yield: 89%) as a cololess oil.

Under hydrogen atmosphere, 1 mL of a methanol suspension containing 87mg of dibenzyl ester-11 and 5% Pd/C was stirred for 5 hours at roomtemperature. The reaction mixture was filtered, and to the filtrate wasadded water, then extracted with hexane. The aqueous portion wascollected, and concentrated under reduced pressure to obtain a solidresidue. The residue was washed with ethyl ether to obtain 31 mg of thefollowing product of(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine [L-I].

L-I: colorless prisms

m.p.: 200° C. (decomposed)

[α]_(D) ^(23.2)=+39.4 (c 1.00, H₂O)

IR (KBr) ν cm⁻¹: 3106, 1621, 1530, 1476, 1393.

¹H-NMR (400 MHz, D₂O)δ: 2.72 (1H, m), 2.80 (1H, dd, J=15.0, 7.8 Hz),3.81 (1H, d, J=10.7 Hz).

¹³C-NMR (100.6 MHz, D₂O)δ: 31.3 (dd, J=11.6, 8.6 Hz), 34.1 (br), 54.2(br), 113.7 (dd, J=288.2, 286.1 Hz), 172.7, 173.7.

¹⁹F-NMR (376.5 MHz, D₂O)δ: −71.4 (1F, dd, J=157.6, 13.6 Hz), −69.0 (1F,dd, J=157.2, 14.8 Hz).

[Pharmacological Experiments] (1) Action on metabotropic L-glutamatereceptors (mGluR) of Group-I

Evaluation of depolarization activity

In accordance with Shinozaki's method [Shinozaki et al., Br. J.Pharmacol., 98, 1213-1224(1989)], the depolarization activities of theabove-prepared compounds were measured using spinal specimen enucleatedfrom neonatal rats.

With respect to each of the compounds of the invention [hydrochloridesof L-I to IV, and D-I to IV] and optical isomers of CCG[2-(carboxycyclopropyl)glycine, which was a known agonist for mGluR],the measurement was carried out in the following manner.

Under perfusing an artificial cerebrospinal fluid including 0.5 μM oftetrodotoxin, depolarization at the ventral root of the spinal cord wascaused by the test compound, and extracellularly recorded. Themeasurement was repeated in the range of 10⁻³ to 10⁻⁷ M of the testcompound, and thereby the minimal effective concentration (MEC) wasestimated.

The activity of each compound was determined on the basis of the ratioof the MEC of the test compound to that of the corresponding opticalisomer of COG. The results are set forth in Table 1.

TABLE 1 Ratio of the activity of each compound based on that of thecorresponding optical isomer of CCG I II III IV L 3 0.3 2 1.5 D 1.5 0.1<0.1 <0.01

TABLE 2 Minimal effective concentration (μM) of the correspondingoptical isomer of CCG I II III IV L 2 200 100 0.3 D 3 0.1 300 5

When 3×10⁻⁵ M of an NMDA antagonist (CPP[3-{(RS)-2-carboxypiperazin-4-yl}propyl-1-phosphoric acid] or D-AP5[D(−)-2-amino-5-phosphonovaleric acid]) was added to the artificialcerebrospinal fluid, it was confirmed that the depolarization activitiesof L-I and L-II are hardly affected by the NMDA antagonist.

It was also confirmed that L-I causes NMDA-like depolarization at themagnitude of approx.1/33 based on that caused by known DCG-IV[(2S,1′R,2′R,3′R)-2-(2,3-dicarboxycyclopropyl)glycine]. This means thatthe depolarization caused by L-I is practically negligible.

When 100 μM of an NMDA antagonist (D-AP5) and 100 μM of a non-NMDAantagonist (CNQX [6-cyano-7-nitroquinoxaline-2,3-dione]) wereincorporated into the artificial cerebrospinal fluid, it was confirmedthat the depolarization activities of L-I and -II are hardly reduced bythese antagonists although those of L-III, L-IV and D-I to D-IV areinhibited.

Furthermore, when 1 mM of an antagonist for mGluR of Group I [MCPG,(RS)-α-methyl-4-carboxyphenylglycine] was incorporated into theartificial cerebrospinal fluid, it was confirmed that the depolarizationactivities of L-I and L-II are inhibited by the antagonist.

(2) Action on metabotropic L-glutamate receptors(mGluRs) of Group-II

Evaluation of the inhibitory activity on monosynaptic reflex

In accordance with Ohtsuka's method [Ohtsuka M., Seitai no Kagaku,36(4), 325-327], the monosynaptic reflex inhibitory activities of theabove-prepared compounds were measured using spinal cord specimenenucleated from neonatal rats in the following manner.

The spinal cord was enucleated together with the vertebral column from aneonatal Wistar rat anesthetized with ether, and soaked in an artificialcerebrospinal fluid saturated with a mixed gas of O₂ and CO₂ (95%: 5%).In the artificial cerebrospinal fluid, the spinal cord in the vertebralcolumn was sectioned under a microscope to prepare a spinal cordspecimen having the ventral and dorsal roots of L3 to L5. The preparedsample was set in the perfusion system, and then the artificialcerebrospinal fluid saturated with the above mixed gas was made to flow.

The test compound was added into the perfusion liquid, and themonosynaptic reflex was measured by the steps of giving a pulse to thedorsal root through an suction electrode, recording the reflex potentialat the ventral root, and observing a rapidly responding spike(corresponding to the monosynaptic reflex) and delayed depolarizationwith nonsynchronous potential drift. The measurement was repeated exceptthat the concentration of the test compound was varied, and thereby theminimal effective concentration (MEC) was estimated.

This experiment revealed that the MEC of L-CCG-I (a known agonist formGluR) is 0.2 μM and that the cyclopropylglycine derivative L-I of theinvention inhibits the spinal monosynaptic reflex three times as strongas L-CCG-I.

Further, the measurement was repeated except that 0.3 mM of anantagonist for mGluR of Group II [MCCG-I, (2S,3S,4S)-α-methyl-2-(2-carboxycyclopropyl)glycine] was incorporated into theartificial cerebrospinal fluid. By this measurement, it was confirmedthat the inhibitory activities on the monosynaptic reflex of L-I andL-II are vanished by the antagonist.

(3) Summary of pharmacological actions belonging to cyclopropylglycinederivatives of the invention

1) The depolarization activity of L-I was slightly (5%) inhibited by 100μM of D-AP5 (NMDA antagonist), hardly inhibited by CNQX (non-NMDAantagonist), and completely inhibited by 1 mM of MCPG (antagonist formGluR of Group I).

Further, L-I reduced the spinal monosynaptic reflex, but this action wasinhibited by MCCG (antagonist for mGluR of Group II).

Those results of the experiments indicate that L-I works as an agonistfor mGluRs of both Groups-I and -II, and the minimal effectiveconcentration (MEC) suggests that the agonistic effect for Group-II ispredominant to that for Group-I.

2) L-II was insensitive to CNQX (non-NMDA antagonist), and hence ispresumed to be an agonist for mGluR of Group-I.

3) L-III, L-IV and all D-isomers are presumed to be NMDA agonists.

4) L-III lacks the activity for inhibiting the transporter for glutamicacid although L-CCG-III has that activity. [Folia Pharmacol. Jpn., No.104, 177-187 (1994); Br. J. Pharmacol., 98, 1213-1224(1989)].

INDUSTRIAL APPLICABILITY

The cyclopropylglycine derivative of the invention, particularly(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine, hasexcellent characteristics as an agonist for metabotropic L-glutamatereceptors, and is therefore supposed to be used as a sedative, ananalgesic, an anesthetic enhancer, an anticonvulsant, and a remedy forvarious cerebral functional disorders (e.g., Huntington's disease,epilepsy, Parkinson's disease) caused by degeneration or ischemic deathof neurons.

Further, the cyclopropylglycine derivative of the invention is usablefor pharmaceutical studies to develop antagonists for L-glutamic acidreceptors.

What is claimed is:
 1. A cyclopropylglycine derivative having thefollowing formula:

in which each of R¹ and R² independently represents a hydroxyl group oran alkoxy group having 1 to 6 carbon atoms, each of R³ and R⁴independently represents a hydrogen atom or an alkyl group having 1 to 6carbon atoms, and each of X¹ and X² independently represents a halogenatom.
 2. The cyclopropylglycine derivative of claim 1, wherein each ofR³ and R⁴ represents a hydrogen atom, and each of X¹ and X² represents afluorine atom. 3.(2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine.
 4. Anagonist for metabotropic L-glutamate receptors comprising, as an activecompound, a cyclopropylglycine derivative having the following formula:

in which each of R¹ and R² independently represents a hydroxyl group oran alkoxy group having 1 to 6 carbon atoms, each of R³ and R⁴independently represents a hydrogen atom or an alkyl group having 1 to 6carbon atoms, and each of X¹ and X² independently represents a halogenatom.
 5. The agonist for metabotropic L-glutamate receptors of claim 4wherein each of R³ and R⁴ represents a hydrogen atom, and each of X¹ andX² represents a fluorine atom.
 6. An agonist for metabotropicL-glutamate receptors comprising (2S,1′S,2′S)-2-(2′-carboxy-3′,3′-difluoro)cyclopropylglycine as an active compound.
 7. A lactamderivative having the following formula:

in which R² represents a hydroxyl group or an alkoxy group having 1 to 6carbon atoms, R³ represents a hydrogen atom or an alkyl group having 1to 6 carbon atoms, and each of X¹ and X² independently represents ahalogen atom.